1. Field of the Invention
One aspect of this invention relates to compositions of matter which provide the components required for increasing the intracellular synthesis of adenosine 5-triphosphate (hereinafter ATP) and to methods for increasing ATP levels in skeletal muscle cells. More particularly, the present invention relates to compositions and methods which increase ATP levels and physical performance levels when administered orally and to pharmaceutical preparations which increase the rate of wound repair and/or which have an antimicrobial effect when applied locally.
2. The Role of ATP in Cell Metabolism
The chemistry and biology of the adenine nucleotides began in 1847 with their isolation by Liebig (Liebig's ann 62: 317, 1847). At first these compounds were thought to be only degradation components of the nucleic acids; however, it was later realized that the chemistry and biology of the nucleotides derive from the purine bases adenine and hydroxanthine to which the five-carbon sugar d-ribose is added to form biologically active nucleoside precursors. The addition of phosphate to a nucleoside (purine base+pentose+phosphate) forms a nucleotide. By the early 1940's, attempts were being made to utilize the nucleotide ATP in the form of a Ca or Mg salt for energy enhancement.
ATP plays a diverse role in intermediary cell metabolism due to its high energy phosphate bonds. This was first demonstrated by Lipmann with regard to ATP's role in glycolysis (Advances in Enzymology 1, 99, 1947). Within the next four years, the central role ATP plays in respiration, carbohydrate metabolism, fat metabolism and the synthesis of urea and glutamine was clearly established.
Energy for muscular contraction and hence work comes from the hydrolysis of ATP. In skeletal muscle, ATP is hydrolized to adenosine diphosphate (hereinafter ADP) by the enzyme myosin ATPase. The hydrolysis of ATP to ADP is accompanied by the release of energy, and the process may be represented by the following reaction: ##STR1##
Muscular contraction proceeds for a few seconds during this liberation of energy.
ATP blood levels are only reported to decrease over short time periods. For example, ATP blood levels have been observed to drop in humans and horses during periods of highly intensive exercise. It is apparent that if contractile activity is to be maintained during such exercise, rapid synthesis of ATP must occur intracellularly (Hodgson, Equine Practice, Vet Clinics, December 1985).
There are two processes which provide intracellular replenishment of ATP: oxidative (aerobic) phosphorylation of substrates from circulating fatty acids and glucose and intramuscular glycogen and triglycerides; and, anaerobic phosphorylation in which ATP is generated from creatine phosphate, circulating glucose and glycogen stores.
In oxidative phosphorylation, muscle cells are unable directly to obtain ATP circulating in the blood; the reason appears to be associated with the binding of ATP's triphosphate component to the cell membrane. In most cells, the fewer the phosphates attached to the nucleotide, the more readily the nucleotide will be absorbed by the cell. Thus, cells will generally absorb adenosine monophosphate (hereinafter AMP) faster than ADP, and they will absorb ADP faster than ATP. AMP or ADP which has been absorbed by the cell must be rephosphorylated intracellularly to form ATP. This process involves the passage of hydrogen molecules from one step to the next along a reaction chain with the concurrent release of large amounts of chemical energy. Approximately half of this energy is utilized by the cell for further rephosphorylation of AMP or ADP, and the balance is given off as heat energy.
At the onset of exercise or under conditions where oxygen transport is insufficient, ATP stores built by oxidative phosphorylation become depleted; ATP, however, must still be made available to provide energy for muscular contraction. Anaerobic phosphorylation mechanisms are utilized by the muscle cells to provide the required ATP under these circumstances. Anaerobic phosphorylation occurs solely in the cell cytosol and mitochondria and involves high energy phosphates (phosgenes) represented by creatine phosphate (hereinafter CP), ADP and AMP.
The most important anaerobic phosphorylation mechanism appears to involve CP and may be represented as follows: ##STR2##
A second mechanism for restoring ATP levels in muscle cells is the myokinase reaction. Through this mechanism, muscle cells condense two molecules of ADP to form one molecule of ATP and one molecule of AMP. The mechanism may be represented as follows: ##STR3##
Substrate utilization through oxidative phosphorylation in, for example, exercising humans or animals such as racehorses, depends on the intensity of the work. ATP contributions through oxidative phosphorylation are directly related to the pace and speed of muscular contraction. As exercise continues, ATP becomes depleted and is restored by donations of energy from CP. When CP is depleted, other stores of energy are required. Although the myokinase reaction is present in skeletal muscle, it may have only a limited role in energy metabolism. Glycolysis and its end product pyruvate, which may be converted to lactate, provide the ongoing energy supply. In horses, the glycolytic process reaches peak efficiency approximately thirty seconds from the onset of exercise. Because equines have a large store of glycogen, this substrate is able to provide a considerable source of energy during exercise.
3. The Role of ATP in Wound Repair
The ATP dependency of the contractile mechanism in both striated and smooth muscle cells is signaled by the presence of divalent cations, particularly Ca++ and Mg++. It is significant that the content and function of calcium and magnesium in isolated myofibroblasts are analogous to their content and function in smooth muscle cells; hence, it appears that the contractile mechanism of the myofibroblasts is also dependent on ATP (Science, 1644-48, March 1987).
The myofibroblasts are a specialized population of fibroblasts; fibroblasts are connective tissue cells which, when differentiated, form binding and supporting connective tissue (e.g. collagen) such as tendons. The myofibroblasts are atypical fibroblasts which combine some of the ultrastructural features of fibroblasts and smooth muscle cells. The myofibroblasts have a dense collection of microfilament bundles that are rich in actin filaments. These bundles are muscle-like contractile fibrils.
A positive correlation has been established between the rate of wound repair in animals and the number of myofibroblasts present at the wound site. In many instances a wound lacks sufficient vascular development to support the nutritional needs of repair processes. This is so even when additional nutrients are provided by intravenous adminstration, since the primary means these nutrients have for reaching the repair site is by diffusion from the vascularized regions adjacent to the wound. An inadequate regenerative capacity of the host is particularly acute where the wound surface to be closed by granulation is large. In such instances, contraction of the wound surface, characterized by the movement of intact dermis over the wound site, plays an important role in the repair process by lessening the size of the wound gap (J. Cenat., 89, 114-123, 1955). It is myofibroblasts, with their muscle-like contractile fibrils, which produce such wound contraction.
As noted above, the contractile mechanism of these fibrils is dependent on ATP. Thus, the rate of localized wound contraction produced by the myofibroblasts is dependent on the amount of ATP available to them as an intracellular energy source. Moreover, ATP serves as an energy source for other wound repair processes, including granulation of the wound by fibroblasts, gluconeogenesis and protein synthesis, and epithelialization.
Also important in the process of wound healing is the prevention or abatement of infection in a wound. Hence, antimicrobial agents have been recognized as important adjuvants to wound healing regimens.
In view of the diverse role ATP plays in cell metabolism and the importance of ATP to overall animal biochemistry and physiology, it is an aim of this invention to provide a method and composition of matter which supplies the components required for increasing the intracellular synthesis of ATP. It is also an aim of this invention to provide a method and composition of matter that exerts antimicrobial activity in a wound and thereby contributes to acceleration of the wound healing process.